Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date nearly 250 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). Two common POLG mutations found in patients with such mitochondrial diseases generate T251I and P587L amino acid substitutions in PolG. Rarely reported independently, the T251I and P587L mutations are usually found in cis. To understand whether T251I or P587L is the primary pathogenic allele or whether both mutations are required to cause disease, we overexpressed and purified WT, T251I, P587L and T252I/P587L double variant forms of recombinant PolG. Whereas each of the single mutation variants displayed approximately half of the DNA polymerase activity of the WT enzyme in steady state kinetics analyses in vitro, the double mutant form was severely compromised and retained only 4% of the WT polymerase activity. In agreement, both DNA polymerase processivity and exonuclease activity were also diminished by the single mutations and severely reduced in the double mutant form. Possible causes of this impaired efficiency of DNA synthesis are reduced affinity to DNA, impaired thermostability, low catalytic efficiency and/or failure to bind the p55 accessory subunit. Functional assays definitively show no defects in binding isotherms for p55 and each p140 variant, which indicates inter-subunit affinities similar to WT. Conversely, data shows reduced DNA binding affinity as expressed by the fraction of DNA bound to the variant forms of p140. This appears to be caused by dramatically compromised thermostability of the p140 variants. In conclusion, our biochemical analyses suggest both T251I and P587L substitutions functionally impair PolG, with greater pathogenicity predicted for the single P587L variant. In combination, T251I and P587L have extreme thermal lability leading to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients. In the POLG2 gene, we describe an extended Belgian pedigree where seven individuals presented with adult-onset cerebellar ataxia, axonal peripheral ataxic neuropathy and tremor, in variable combination with parkinsonism, seizures, cognitive decline, and ophthalmoplegia. We sought to identify the underlying molecular etiology and characterize the mitochondrial pathophysiology of this neurological syndrome. Clinical, neurophysiological and neuroradiological evaluations were conducted. Patient muscle and cultured fibroblasts underwent extensive analyses to assess mitochondrial function. Genetic studies including genome-wide sequencing were conducted. Hallmarks of mitochondrial dysfunction were present in patients tissues including ultrastructural anomalies of mitochondria, mosaic cytochrome c oxidase deficiency, and multiple mtDNA deletions. We identified a splice acceptor variant in POLG2, c.970-1G>C, segregating with disease in this family and associated with a concomitant decrease in levels of POLG2 protein in patient cells. This work extends the clinical spectrum of POLG2 deficiency to include an overwhelming, adult-onset neurological syndrome that includes cerebellar syndrome, peripheral neuropathy, tremor, and parkinsonism. We therefore suggest to include POLG2 sequencing in the evaluation of ataxia and sensory neuropathy in adults, especially when it is accompanied by tremor or parkinsonism with white matter disease. The demonstration that deletions of mtDNA resulting from autosomal dominant POLG2 variant lead to a monogenic neurodegenerative multi-component syndrome, provides further evidence for a major role of mitochondrial dysfunction in the pathomechanism of non-syndromic forms of the component neurodegenerative disorders.